: The primary cilium is a tiny, immotile antenna-shaped protrusion found on nearly every cell in the human body. This organelle is essential for organ development and function, serving as a hub for several critical cell signaling pathways. Perturbing cilia instigates a slew of ciliopathies that include developmental, sensory, metabolic, and regenerative disorders. While the biological roles of the cilium are known, it is unclear at a biochemical level how or why the cilium is needed for its constituent cascades to operate. This prevents an understanding of how the cilium serves any of its biological functions. It also stymies our ability to control ciliary signaling therapeutically. The goal of the work proposed here is to understand the biochemical and biophysical principles governing ciliary signal transduction. Rather than relying on conventional genetic and cell biological approaches in the field, we are harnessing non-traditional biochemical and physiologic tools from other areas of biology; this includes reconstitution systems and live-cell sensors for key transduction events. This proposal uses the Hedgehog (Hh) pathway, a model ciliary cascade and fundamental regulator of embryogenesis and stem cell biology, to address these outstanding questions. At the ciliary membrane, the seven-transmembrane (7TM) protein Smoothened (SMO) is the pivotal molecule controlling essentially all the Hh pathway?s biological activities. The following three projects will together uncover the molecules and mechanisms by which SMO and related ciliary 7TM proteins signal: 1) What are biochemical mechanisms that regulate SMO activation? This builds on the recent discovery that ion gradients and membrane lipids are key regulators of the crucial initial steps of Hh signaling. These studies now provide a platform to investigate how membrane cholesterol, a candidate ?messenger? linking upstream pathway events to SMO activity, binds to and activates SMO. These efforts have also revealed additional SMO regulatory factors, which we will identify biochemically. 2) How does SMO, once activated, communicate to GLI transcription factors that control expression of Hh pathway target genes? We will pinpoint SMO?s long-sought immediate downstream effector in the Hh pathway and test the hypothesis that the cilium serves primarily as a ?meeting place? to concentrate activated pathway components together. 3) How do these regulatory influences operate within the cilium? These experiments will test the physiological significance of the regulatory factors we identify biochemically, as well as quantitatively compare and selectively manipulate signal transduction in the ciliary and ?cell body? compartments. Each project also provides stepping stones to investigate related mechanisms that govern 7TM proteins in many other ciliary signaling cascades. The techniques deployed and principles learned from the Hh cascade will thus be broadly applicable to other ciliary pathways. Defining these cascades at a mechanistic level is essential for the development of diagnostic and therapeutic tools for a range of devastating disorders; this includes congenital defects, malignancies, and dysfunction of the nervous, cardiovascular, and excretory systems.